Self-Assembling Cell Aggregates and Methods of Making Engineered Tissue Using the Same

ABSTRACT

A composition comprising a plurality of cell aggregates for use in the production of engineered organotypic tissue by organ printing. A method of making a plurality of cell aggregates comprises centrifuging a cell suspension to form a pellet, extruding the pellet through an orifice, and cutting the extruded pellet into pieces. Apparatus for making cell aggregates comprises an extrusion system and a cutting system. In a method of organ printing, a plurality of cell aggregates are embedded in a polymeric or gel matrix and allowed to fuse to form a desired three-dimensional tissue structure. An intermediate product comprises at least one layer of matrix and a plurality of cell aggregates embedded therein in a predetermined pattern. Modeling methods predict the structural evolution of fusing cell aggregates for combinations of cell type, matrix, and embedding patterns to enable selection of organ printing processes parameters for use in producing an engineered tissue having a desired three-dimensional structure.

FIELD OF THE INVENTION

The present invention relates generally to the field of tissueengineering and more particularly to production of engineered tissueshaving desired structures.

BACKGROUND

Tissue engineering provides promising solutions to problems caused bythe growing demand for organ/tissue replacement therapies coupled with achronically low supply of transplantable organs. In the United States,for example, thousands of people are on the national waiting list fororgan transplants. Many will likely perish for lack of suitable organreplacements. To lessen and eventually solve the problem of inadequatesupply of organs for transplantation, tissue engineers need to be ableto build and grow transplantable organs or organ substitutes in alaboratory, with high precision, on large scale, and in a relativelyshort amount of time.

A variety of methods and devices for tissue engineering have beenattempted and developed with limited success. There has been somesuccess, for example, with production of non-vascularized tissues (e.g.,cartilage and tendons). However, assembly of vascularizedthree-dimensional soft organs has not been accomplished.

One of the more promising tissue engineering technologies that isemerging is organ printing. Organ printing is generally acomputer-aided, dispenser-based, three-dimensional tissue-engineeringtechnology aimed at constructing functional organ modules and eventuallyentire organs layer-by-layer. Organ printing technology, prior to thepresent invention, has been based on seeding individual cells intobiodegradable polymer scaffolds or gels, similar to traditional tissueengineering approaches, but using a dispensing apparatus that employs,for example, a technology analogous to an ink-jet printer or morecomplex three-dimensional rapid prototype printers (which partiallyexplains the origin of the phrase “organ printing”). Once implanted inthe scaffold, the embedded cells are cultured in a bioreactor forseveral weeks during which time the cell population expands. Theresulting tissue may be implanted into a patient where the maturation ofthe new organ may or may not take place.

Organ printing based on deposition of single cells in a scaffold hasmany of the same shortcomings as traditional tissue engineering. First,it has not yet achieved production of tissues that require provision ofa vascular network, which limits the size and type of the tissue thatcan be produced. It is also difficult to control the structure of thetissue as it grows from the seeds. Thus, tissue having the desired shapeand required stability for a target organ cannot be produced reliably.Further, the individually seeded cells may not survive long enough tosufficiently proliferate. After the cells have been seeded in a scaffolda relatively long incubation period is also required to allow the cellsenough time to multiply and form a significant amount of tissue.

Therefore, what is needed is a new and improved technology that enablesrapid, reliable, and precise building of target organs. What is furtherneeded is a method, combined with appropriate devices, capable ofproducing mechanically stable and long-lived three-dimensionalorganotypic tissue structures.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a method of producinga plurality of fused aggregates forming a desired three-dimensionalstructure. The method comprises depositing a layer of a matrix on asubstrate; embedding a plurality of cell aggregates, each comprising aplurality of cells, in the layer of the matrix, the aggregates beingarranged in a predetermined pattern; allowing at least one aggregate ofsaid plurality of cell aggregates to fuse with at least one otheraggregate of the plurality of cell aggregates to form the desiredstructure; and separating the structure from the matrix.

Also provided is a computerized method of producing a plurality of fusedaggregates forming a desired three-dimensional structure. The methodcomprises selecting an embedding pattern by which a plurality of cellaggregates are to be embedded in a matrix, the plurality of cellaggregates each comprising a plurality of cells; establishing a group ofcandidate matrices to be evaluated for use as the matrix based at leastin part on the compatibility of the candidate matrices with the cellaggregates; executing a computer simulation based on parameterscomprising an interaction force between two of the cells of each of theplurality of cells, an interaction force between one of the cells andeach of the candidate matrices, and an interaction force between twovolume elements of each of the candidate matrices to predict structuralevolutions of the pluralities of cells that are likely to occur if saidplurality of cell aggregates are embedded in each of the candidatematrices in accordance with the embedding pattern; and selecting one ofthe candidate matrices that is predicted to result in the cellaggregates evolving into the desired three-dimensional structure for usein producing fused aggregates forming the desired structure.

Also provided is a computerized method of producing a plurality of fusedaggregates forming a desired three-dimensional structure. The methodcomprises identifying a matrix to be used to temporarily support aplurality of cell aggregates embedded therein in accordance with apredetermined embedding pattern, the cell aggregates each comprising aplurality of cells; establishing a group of candidate cell types to beevaluated for use as a constituent of at least one of the plurality ofcell aggregates based at least in part on the suitability of cells ofthe candidate cell types to perform a desired biological function;executing a computer simulation based on parameters comprisinginteraction forces between two cells of the same type for each of thecandidate cell types, an interaction force between one of the cells ofeach candidate cell type and the matrix, and an interaction forcebetween two volume elements of the matrix to predict structuralevolutions of the pluralities of cells that are likely to occur for eachcell type if said plurality of cell aggregates comprising cells of therespective candidate cell type are embedded in the matrix in accordancewith the embedding pattern; and selecting one of the candidate celltypes that is predicted to result in the cell aggregates evolving intothe desired three-dimensional structure for use in producing fusedaggregates forming the desired structure.

Also provided is a computerized method of producing a plurality of fusedaggregates forming a desired three-dimensional structure. The methodcomprises identifying a matrix to be used to temporarily support aplurality of cell aggregates embedded therein in accordance with anembedding pattern, the cell aggregates each comprising a plurality ofcells; establishing a group of candidate embedding patterns to beevaluated for use as the embedding pattern by which said plurality ofcell aggregates are to be embedded in the matrix based at least in parton similarity of the embedding pattern to the desired three-dimensionalstructure; executing a computer simulation based on parameterscomprising interaction forces between two of the cells, an interactionforce between one of the cells and the matrix, and an interaction forcebetween two volume elements of the matrix to predict structuralevolutions of the pluralities of cells that are likely to occur for eachof the candidate embedding patterns if said plurality of cell areembedded in the matrix in accordance with the respective embeddingpattern; and selecting one of the candidate embedding patterns that ispredicted to result in the cell aggregates evolving into the desiredthree-dimensional structure for use in producing fused aggregatesforming the desired structure.

In another embodiment, the present invention provides a compositioncomprising a plurality of cell aggregates, wherein each cell aggregatecomprises a plurality of living cells, and wherein the cell aggregatesare substantially uniform in size and/or shape.

The present invention also provides a method of preparing a plurality ofcell aggregates. The method comprises preparing a cell suspensioncomprising a plurality of living cells; centrifuging the cell suspensionto form a cellular material comprising at least some of the plurality ofliving cells; extruding the cellular material through an orifice; andforming the extruded cellular material into cell aggregates ofsubstantially uniform size or shape.

Also provided is an apparatus for producing a plurality of substantiallyuniform cell aggregates. The apparatus comprises an extrusion systemadapted to receive a container holding a pellet comprising a pluralityof cells and to extrude the pellet through an orifice; and a cuttingsystem operable to cut the extrudate into a plurality of pieces as theextrudate is being extruded through the orifice.

The present invention also provides a three-dimensional layeredstructure. The structure comprises at least one layer of a biocompatiblematrix; and a plurality of cell aggregates, each cell aggregatecomprising a plurality of living cells; wherein the cell aggregates areembedded in the at least one layer of biocompatible matrix in apredetermined pattern.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective of one embodiment of apparatus for making cellaggregates of the present invention;

FIG. 2 is an end view of the apparatus;

FIG. 3 is a side elevation thereof;

FIG. 4 is a top plan view thereof;

FIG. 5 is an enlarged fragmentary perspective of the apparatus;

FIGS. 6 and 7 are perspectives of the apparatus showing a sequence ofoperation of a cutting system;

FIGS. 8A-8L show initial and final cell aggregate configurations forsimulations (FIGS. 8A-8B and 8K-8L) and experiments embedding CHO cellaggregates in Neurogel™ disks (FIGS. 8C-8D), and in collagen gels ofconcentration 1.0 mg/ml (FIGS. 8E-8F), 1.2 mg/ml (FIGS. 8G-8H), and 1.7mg/ml (FIGS. 8I-8J), as described in Example 2;

FIG. 9A shows the boundary between two adjacent aggregates in a ringstructure (FIGS. 9B and 9C) embedded in 1.0 mg/ml collagen gel atvarious times, as described in Example 2;

FIG. 10 is a plot of the angle between the tangents to the boundaries ofthe adjacent aggregates vs. time, as described in Example 2;

FIGS. 11A-11B are plots (at different scales) of total interactionenergy vs. number of Monte Carlo steps, for the simulation in Example 3;

FIGS. 12 and 13 are sequences of modeling images showing the simulationof ring structure formation (FIG. 12) and tube formation (FIG. 13), asdescribed in Example 4;

FIGS. 14 and 15 are plots of B_(cg) vs. number of Monte Carlo steps fora simulation of ring structure formation (FIG. 14) and tube formation(FIG. 15), as described in Example 4;

FIG. 16 is a fluorescent image showing ring structure formation whenfluorescently labeled cell aggregates are embedded in agarose gel, 1.7mg/ml collagen gel, and 1.0 mg/ml collagen gel, an image of the fusionof two neighboring aggregates embedded in 1.0 mg/ml collagen gel, and anenlarged view of the contact region between two fused aggregatesembedded in 1.0 gm/ml collagen, as described in Example 5;

FIGS. 17A-17I is a sequence of fluorescent images showing tube structureformation over time, as described in Example 5; and

FIG. 18 is an image of CHO cell aggregates of approximately 500 microndiameter, produced as described in Example 1, printed in a hexagonpattern on 1.0 mg/ml collagen.

Corresponding reference characters indicate corresponding partsthroughout the drawings.

DETAILED DESCRIPTION

In contrast to previously known methods of tissue engineering which arebased on seeding individual cells into biodegradable polymer scaffoldsor gels, the methods of the present invention use cell aggregates as thebuilding blocks of tissue formation. Using cell aggregates as buildingblocks instead of isolated cells has a number of advantages. Forexample, because cell aggregates may be composed of hundreds tothousands of cells (i.e., they are essentially a prefabricated piece oftissue), cell aggregates provide critical cell concentrations, which maybe difficult to achieve by other methods, and can significantly reducethe time required to “print” the desired structure or tissue.Furthermore, the mechanical hardship involved in the dispensing process(e.g., in the ink-jet technology to print individual cells) is lessdamaging to cell aggregates, which contain a multitude of cells, than itis for individual cells which may be damaged or killed during thedispensing process. In addition, because cell aggregates are alreadythree-dimensional tissue fragments, they are more amenable to fusing toform a three-dimensional structure, than are single cells. Finally,because cell aggregates may contain several cell types and a pre-builtinternal structure, considerable time can be saved during post-processtissue and organ maturation.

One process of making an engineered tissue having a desired structure(e.g., an organotypic tissue) using the inventive methods and materialsdescribed herein involves use of a composition (sometimes referred to as“bio-ink”) comprising a plurality of cell aggregates (sometimes referredto as “bio-ink particles”). A target tissue structure blueprint (e.g., aseries of images showing the target tissue structure (e.g., organ) inthin sections which may be reconstituted, for example by a computer,into a three-dimensional representation of the tissue structure) is alsoacquired. The blueprint may be obtained, for example, by MRI. Then adispensing unit under the guidance of a computer control unit embeds thecell aggregates into a matrix (e.g., a bio-degradable polymeric or gelscaffold) according to a geometric pattern that is designed to result inthe cell aggregates evolving into a structure representative of thetarget organ. The cell aggregates reside in the matrix where they fusetogether and evolve into an organotypic tissue structure. The tissuestructure is separated from the matrix (e.g., by exposing the matrix toa stimulus that degrades the matrix). Then the engineered tissue isplaced in a bio-reactor/maturation unit for accelerated maturation andpost-processing support.

The present invention recognizes that cell aggregates (e.g., bio-inkparticles) have the ability to fuse into three-dimensional organotypicstructures upon deposition into a stimuli-sensitive matrix made ofbiodegradable polymers or gels. The ability of cell aggregates to fuseis based on the concept of tissue fluidity, according to which embryonictissues (or more generally, tissues composed of adhesive and motilecells) in many respects can be considered as liquids. In particular, insuspension or on non-adhesive surfaces, various multicellular aggregates(comprising adhesive and motile cells) round up into spherical shapesimilarly to liquid droplets. Thus, closely-spaced (e.g., within about0.5 aggregate diameters) or contiguously-placed aggregates embedded in amatrix formed from appropriately chosen gels or polymers can fuse toform tissue constructs having a desired geometry. Moreover, theprinciples of the Differential Adhesion Hypothesis (M. S. Steinberg,“Does differential adhesion govern self-assembly processes inhistogenesis? Equilibrium configurations and the emergence of ahierarchy among populations of embryonic cells”, J Exp Zool 173, p.395-433 (1970); M. S. Steinberg and T. J. Poole, “Liquid behavior ofembryonic tissues”, in Cell Behavior, pp. 583-697, (eds. Bellairs, R.,Curtis, A. S. G. & Dunn, G.) Cambridge University Press (1982)) havebeen determined to predictably describe evolution of the structureformed by constituent cells of fusing cell aggregates.

Having provided a general overview of one embodiment of a process ofproducing tissue, systems, processes and materials used therein will nowbe described in more detail.

Bio-Ink

The present invention thus provides compositions comprising cellaggregates (i.e., “bio-ink” or “bio-ink compositions”) for use in themethods described herein. These compositions comprise a plurality ofcell aggregates, wherein each cell aggregate comprises a plurality ofliving cells, and wherein the cell aggregates are substantially uniformin size and/or shape. The cell aggregates are characterized by thecapacity: 1) to be delivered by computer-aided automatic celldispenser-based deposition or “printing,” and 2) to fuse into, orconsolidate to form, self-assembled histological constructs.

In contrast to cell aggregates produced by previously known methods thatmay vary in both size and shape, the bio-ink of the present inventioncomprises a plurality of cell aggregates that have a narrow size andshape distribution (i.e., are substantially uniform in size and/orshape). The uniformity of the cell aggregates in the bio-ink isparticularly advantageous when the bio-ink used in the methods describedherein, since the uniformity of shape and/or size of the cell aggregatesprovides for greater ease in printing and more predictability inaggregate fusion.

Thus, in one embodiment, the bio-ink comprises a plurality of cellaggregates, wherein the cell aggregates are substantially uniform inshape. By “substantially uniform in shape” it is meant that the spreadin uniformity of the aggregates is not more than about 10%. In anotherembodiment, the spread in uniformity of the aggregates is not more thanabout 5%.

The cell aggregates used herein can be of various shapes, such as, forexample, a sphere, a cylinder (preferably with equal height anddiameter), rod-like, or cuboidal (i.e., cubes), among others. Althoughother shaped aggregates may be used, it is generally preferable that thecell aggregates be spherical, cylindrical (with equal height anddiameter), or cuboidal (i.e., cubes), as aggregates of these shapes tendto cause less clogging of the dispensers during printing.

For example, in one embodiment, the bio-ink comprises a plurality ofaggregates wherein the aggregates are substantially spherical. By“substantially spherical” it is meant that the principle radii ofcurvature of the cell aggregate are substantially equal at all points onthe surface of the aggregate (i.e., vary by about 10% or less over allpoints on the surface of the aggregate). In a preferred embodiment, theprinciple radii of curvature of the cell aggregate vary by about 5% orless over all points on the surface of the aggregate. In contrast,previously known examples of cell aggregates are typically non-uniformspheroidal cell aggregates. By “spheroidal cell aggregates” it is meantthat while the aggregate is generally shaped like a sphere, the radii ofcurvature of the aggregate are not substantially equal for all points onthe surface of the aggregate (i.e., vary by substantially more than 10%over all points on the surface of the aggregate).

Although the exact number of cells per aggregate is not critical, itwill be recognized by those skilled in the art that the size of eachaggregate (and thus the number of cells per aggregate) is limited by thecapacity of nutrients to diffuse to the central cells, and that thisnumber may vary depending on cell type. Cell aggregates may comprise aminimal number of cells (e.g., two or three cells) per aggregate, or maycomprise many hundreds or thousands of cells per aggregate. Typically,cell aggregates comprise hundreds to thousands of cells per aggregate.For purposes of the present invention, the cell aggregates are typicallyfrom about 100 microns to about 600 microns in size, although the sizemay be greater or less than this range, depending on cell type. In oneembodiment, the cell aggregates are from about 250 microns to about 400microns in size. In another embodiment, the cell aggregates are about250 microns in size. For example, spherical cell aggregates arepreferably from about 100 microns to about 600 microns in diameter,cylindrical cell aggregates are preferably from about 100 microns toabout 600 microns in diameter and height, and the sides of cuboidal cellaggregates are preferably from about 100 microns to about 600 microns inlength. Aggregates of other shapes will typically be of similar size.

Although the size of the cell aggregates may vary, it is preferable thatthe size of the aggregates present in a particular bio-ink compositionbe substantially uniform. Compositions comprising aggregates ofsubstantially uniform size are generally more amenable for deposition ofthe aggregates using automated depositing devices. For instance, if thecell aggregates differ in size, it will be difficult to reliably deposita single cell aggregate at a desired position in the matrix. By“substantially uniform in size” it is meant that the aggregates' sizedistribution has a spread not larger than about 10%. In one embodiment,the aggregates' size distribution has a spread not larger than about 5%.

Preferably, the bio-ink comprises cell aggregates that are bothsubstantially uniform in size and substantially uniform in shape.

Many cell types may be used to form the bio-ink cell aggregates. Ingeneral, the choice of cell type will vary depending on the type ofthree-dimensional construct to be printed. For example, if the bio-inkparticles are to be used to print a blood vessel type three dimensionalstructure, the cell aggregates will advantageously comprise a cell typeor types typically found in vascular tissue (e.g., endothelial cells,smooth muscle cells, etc.). In contrast, the composition of the cellaggregates may vary if a different type of construct is to be printed(e.g., intestine, liver, kidney, etc.). One skilled in the art will thusreadily be able to choose an appropriate cell type(s) for theaggregates, based on the type of three-dimensional construct to beprinted. Non-limiting examples of suitable cell types includecontractile or muscle cells (e.g., striated muscle cells and smoothmuscle cells), neural cells, connective tissue (including bone,cartilage, cells differentiating into bone forming cells andchondrocytes, and lymph tissues), parenchymal cells, epithelial cells(including endothelial cells that form linings in cavities and vesselsor channels, exocrine secretory epithelial cells, epithelial absorptivecells, keratinizing epithelial cells, and extracellular matrix secretioncells), and undifferentiated cells (such as embryonic cells, stem cells,and other precursor cells), among others.

The bio-ink particles may be homocellular aggregates (i.e., “monocolorbio-ink”) or heterocellular aggregates (i.e., “multicolor bio-ink”).“Monocolor bio-ink” comprises a plurality of cell aggregates, whereineach cell aggregate comprises a plurality of living cells of a singlecell type. In contrast, “multicolor bio-ink” comprises a plurality ofcell aggregates, wherein each individual cell aggregate comprises aplurality of living cells of at least two cell types, or at least onecell type and extracellular matrix (ECM) material, as discussed below.

When a single aggregate comprises more than one cell type (i.e.,heterocellular aggregate or “multicolor bio-ink”), the cells within theaggregate may “sort out” to form a particular internal structure for theaggregate. The pattern evolution in sorting out is consistent with thepredictions of the Differential Adhesion Hypothesis (DAH), discussed inmore detail herein. The DAH explains the liquid-like behavior of cellpopulations in terms of tissue surface and interfacial tensionsgenerated by adhesive and cohesive interactions between the componentcells. In general, cells will sort out based on differences in theadhesive strength of the cells. For example, cell types that sort to thecenter of a heterocellular aggregate generally have a stronger adhesionstrength (and thus higher surface tension) than cells that sort to theoutside of the aggregate.

Furthermore, when a heterocellular aggregate is composed of cells oftissues that are neighbors in normal development, in the course ofsorting they may recover their physiological conformation. Thus,heterocellular aggregates may comprise a sort of pre-built internalstructure, based on the adhesive and cohesive properties of thecomponent cells, and the environment in which the cells are located.This can be used to build more complex biological structures. Forexample, while building a simple contractile tube, monocolor bioinkcomposed of muscle cells can be used; to build a blood vessel-likestructure, at least two cell types (i.e., endothelial cells and smoothmuscle cells) can be used. By printing bio-ink particles composed ofthese two cell types randomly dispersed in the aggregate, in the courseof postprinting structure formation, these cell types sort and with theright choice of the scaffold, endothelial cells will line the internalstructure of the tube (i.e., lumen), whereas smooth muscle cells willform the outer layer of the tube. The optimal structure can be achievedby varying the composition of the aggregate (e.g., ratio of endothelialto smooth muscle cells), by the size of the aggregate and thecomposition of the scaffold (e.g., possibly different scaffold materialused in the interior and exterior of the printed construct).

In addition to one or more cell types, the bio-ink aggregates canfurther be fabricated to contain extracellular matrix (ECM) material indesired amounts. For example, the aggregates may contain various ECMproteins (e.g., collagen, fibronectin, laminin, elastin, and/orproteoglycans). Such ECM material can be naturally secreted by thecells, or alternately, the cells can be genetically manipulated by anysuitable method known in the art to vary the expression level of ECMmaterial and/or cell adhesion molecules, such as selectins, integrins,immunoglobulins, and cadherins, among others. In another embodiment,either natural ECM material or any synthetic component that imitates ECMmaterial can be incorporated into the aggregates during aggregateformation, as described below.

The bio-ink cell aggregates can be suspended in any physiologicallyacceptable medium, typically chosen according to the cell type(s)involved. The tissue culture media may comprise, for example, basicnutrients such as sugars and amino acids, growth factors, antibiotics(to minimize contamination), etc.

Bio-ink particles described herein can be used in accordance with themethods of the present invention to produce three-dimensional fusedtissue constructs. The bio-ink can be dispensed using any of a varietyof printing or dispensing devices, such as those discussed below.Therefore, it is advantageous to store the bio-ink compositions in thestorage chamber of a container, such as a printer cartridge, that iscompatible with a printing or dispensing device. Thus, in oneembodiment, the present invention provides a printer cartridgecomprising a storage chamber, wherein the bio-ink is stored in thestorage chamber. Because the aggregates of the bio-ink may begin to fusetogether before printing if allowed to sit for long periods of time, itis generally preferable to transfer the bio-ink to the printer cartridgewithin 2 hours, more preferably within 1 hour, and even more preferablywithin 30 minutes of printing to avoid any fusion of aggregates in thebio-ink before printing.

Method of Making Bio-Ink Particles

A variety of methods of making cell aggregates are known in the art suchas, for example, the “hanging drop” method wherein cells in an inverteddrop of tissue culture medium precipitate and aggregate; shaking cellsuspensions in a laboratory flask; and various modifications of thesetechniques. See, e.g., N. E. Timmins, et al., Angiogenesis 7, 97-103(2004); W. Dai, et al., Biotechnology and Bioengineering 50, 349-356(1996); R. A. Foty, et al., Development 122, 1611-1620 (1996); G.Forgacs, et al., Biophys. J. 74, 2227-2234 (1998); K. S. Furukawa, etal., Cell Transplantation 10,441-445 (2001); R. Glicklis, et al.,Biotechnology and Bioengineering 86, 672-680 (2004); and T. Korff, etal., FASEB J. 15, 447-457 (2001). Although such methods can be used toproduce cell aggregates, the aggregates produced by these methods aretypically not substantially uniform in size and/or shape.

The present invention addresses this problem by providing a methodcapable of reproducibly producing cell aggregates that are substantiallyuniform in size and shape. Therefore, in one embodiment, the presentinvention is directed to a method of preparing a plurality of cellaggregates, the method comprising: preparing a cell suspensioncomprising a plurality of live cells; centrifuging the cell suspensionto form a firm cellular material (e.g., a pellet) comprising at leastsome of the plurality of live cells; transferring the firm cellularmaterial to a container (e.g., a micropipette), the container having anorifice; extruding the cellular material through the orifice; andforming (e.g., by slicing or cutting) the extruded cellular materialinto cell aggregates of substantially uniform size and shape as theextruded cellular material exits the orifice.

As discussed above, a large variety of cell types may be used to formthe bio-ink of the present invention. For example, the cell suspensionmay comprise only one cell type (i.e., when monocolor bio-ink is beingprepared), or alternately, may comprise two or more cell types (i.e.,when multi-color bio-ink is being prepared). Furthermore, the cellsuspension may comprise extracellular matrix-like gel.

Once a cell suspension comprising the desired cell type(s) is chosen,the cell suspension can be centrifuged to form a pellet of cellularmaterial. Although the duration and speed of centrifugation is notcritical, centrifugation should generally occur for a period of time andat a speed sufficient so that at least some of the plurality of livecells in the suspension form a relatively firm pellet. Preferably, thepellet is sufficiently firm so that it can be transferred to acontainer, as described below.

After centrifugation, the cellular material collected in the pellet canbe transferred to a container having an orifice, such as a tube,micropipette, or any other suitable container, by any suitable meansincluding, for example, aspiration. The orifice of the containerpreferably has a size and shape similar to the desired size and shape ofthe cell aggregate being produced. For example, if spherical orcylindrical aggregates are being produced, the orifice preferably iscircular, and has a diameter equal to the desired diameter of the cellaggregate. If the aggregate is cuboidal, the orifice is square shapedand has a side length equal to the desired length of the linear side ofthe cube. Thus, in a preferred embodiment, when the aggregate is to bespherical or cylindrical, the orifice is circular, and has a diameter offrom about 100 microns to about 600 microns. Containers with differentshapes and dimensions may readily be selected based on the desired sizeand shape of the cell aggregate being produced.

Once transferred to the container, the cellular material may optionallybe incubated for a period of time during which the cellular materialconforms to the shape of the container. The time and temperature ofincubation will vary with cell type, and may readily be determined byone skilled in the art. Optionally, ECM materials, such as thosedescribed above, may be added to the cellular material prior totransferring the cellular material to the container.

The cellular material may then be extruded through the orifice of thecontainer, and formed into cell aggregates (e.g., by slicing or cuttingthe extruded cellular material) as the extruded cellular material exitsthe orifice. Preferably, the cellular material is sliced at regularintervals, so as to produce cell aggregates of substantially uniformsize and shape. Advantageously, the cellular material is sliced atintervals about equal to the desired size of the cellular aggregate. Forexample, the cellular material may be sliced at a constant interval, theinterval being from about 100 microns to about 600 microns. In oneembodiment, if cylindrical aggregates are being produced, the orifice iscircular, and the extruded cellular material is sliced in intervalscorresponding to the desired height of the cylindrical cell aggregates.The extruded cellular material can then be used as bio-ink particles. Inanother embodiment, if spherical aggregates are being produced, theorifice is circular, and the extruded cellular material is sliced intocylinders so that the height of the cylinder is equal to its diameter.The resulting cylinders may be subjected to further short-termincubation, as discussed below, to form substantially uniform sphericalaggregates,

Any method capable of accurately slicing the extruded cellular materialin the desired intervals may be used herein. Preferably, the extrudedcellular material is sliced using an automatic device, such as thedevice described below. By using such an automated device, uniformity ofaggregate size and/or shape is achieved, in contrast to previously knownmethods of aggregate formation, and the amount of time required toprepare the aggregates is reduced.

If spherical cell aggregates are being prepared, the sliced(cylindrical) cell aggregates may be incubated in any suitable flask,and shaken to produce substantially spherical aggregates. Although thetime and temperature of incubation may vary depending on cell type, theaggregates are typically incubated at 37° C. for about 1 to about 5hours. Any suitable shaking means can be used including, for example, agyratory shaker or a low shear stress vessel, such as the NASA-developedHigh Aspect Ratio Vessel. Preferably the aggregates are shaken at aspeed of about 15-100 revolutions per minute (rpm). The resultingaggregates are substantially spherical.

This method advantageously produces cell aggregates that aresubstantially uniform in both size and shape. Once formed, cellaggregates of similar composition can be suspended in a suitable medium,as described herein, and can be used as bio-ink.

Apparatus for Making Bio-Ink Particles

Although there are various ways to make cell aggregates that aresuitable for use as bio-ink particles, including by hand, withoutdeparting from the scope of this invention, it may be desirable to usean automated system that is capable of producing a large number ofsubstantially uniform cylindrical cell aggregates (i.e., about equal indiameter and within about 10-20 microns of each other in length) at arelatively high rate. It is also desirable to minimize the number ofcells that are killed when a pellet comprising a plurality of cells iscut into pieces.

Referring to FIGS. 1-7, for example, one embodiment of an automateddevice for producing a plurality of cell aggregates is generallydesignated 101. In general the device 101 comprises an extruding system103 operable to extrude a pellet comprising a plurality of cells throughan orifice 119 (FIG. 2) and a cutting system 105 operable to cut thepellet into a plurality of cell aggregates as the pellet is extrudedthrough the orifice. The operations of the extruding system 103 andcutting system 105 are coordinated so the pellet is cut into a pluralityof substantially uniform pieces as the pellet is being extruded throughthe orifice.

In one embodiment, shown in the drawings, the extruding apparatus 103comprises a motor 111 that is drivingly connected to a piston 115. Themotor 111 is operable to advance the piston 115 through a container 117(FIG. 4) that contains the pellet of cells to thereby extrude the pelletthrough the orifice 119 at one end of the container. The cutting system105 of the particular embodiment shown in the drawings comprises areciprocating cutting blade 123 that is operable to cut the pellet intopieces.

The motor 111 of the illustrated embodiment is a stepper motor.Preferably, the operation of the motor 111 is controlled by anelectronic control system 129 (e.g., desktop computer) in electricalcommunication with the motor 111. The electronic control system 129 isonly schematically illustrated in the drawings. The motor 111 ispreferably operable to periodically pause at intervals (e.g., regularintervals) to allow the cutting system 105 the opportunity to sever theextruded portion of the pellet from the part of the pellet that stillremains in the container 117 while the pellet is stationary. Forexample, the control system 129 can direct the motor 111 to operate at asubstantially constant speed for a period (e.g., a period in the rangeof about 1 to about 2 seconds) and then pause for a brief period (e.g.,a period of about 0.5 seconds). It is understood that other actuators(e.g., a servo motor) could be used instead of a stepper motor withoutdeparting from the scope of the invention.

The motor 111 is mounted on a frame 133. The frame 133 comprisesopposing blocks 137 and a plate 139 that holds the blocks in spacedrelation to one another. The motor 111 is mounted on one of the blocks137 and drivingly connected to a threaded shaft 151. The shaft 151passes through an opening 141 in one of the blocks 137 and extends intoan opening 145 in the other of the blocks. Thus, the threaded shaft 151spans the space between the blocks 137. The shaft 151 is rotatable aboutits axis 157 (e.g., rotatably mounted in the openings by one or morebearings) but restrained from translational movement with respect to theframe 133 in the direction parallel to its axis.

A drive block 163 is slideably mounted between the blocks 137 forsliding movement along the axis 157 of the threaded shaft 151. Thethreaded shaft 151 is received in a threaded bore 177 of a drive block163 so that rotation of the threaded shaft drives the movement of thedrive block along the axis 157 of the threaded shaft. The drive block163 is preferably slidably mounted on one or more guide rails that spanthe distance between the blocks 137 and are parallel to the threadedshaft 151. As illustrated in the drawings, for example, the drive block163 can be mounted on four parallel guide rails 169 that are received incorresponding through-holes 173 through the drive block 163 thatarranged in a pattern centered on the threaded bore 177.

A piston 115 (e.g., a stainless steel wire rod) is connected to thedrive block 163 so that sliding movement of the drive block drivesmovement of the piston (e.g., along a longitudinal axis of the piston).It will be understood that various mechanisms may be used to connect thepiston to the drive block, including numerous different arrangements ofgears and/or linkages. In the embodiment shown in the drawings, forexample, the piston 115 is secured to the drive block 163 by a pistonmount 181 on the drive block. The piston mount comprises a clamp 185that is releasably secured (e.g., by screws or other suitable fasteners)to an anvil 189 formed on one side of the drive block 163. A piston 115is received between the clamp 185 the anvil 189 where it is secured tothe piston mount 181 (e.g., by tightening the fasteners to squeeze apiston between the clamp and the drive block). When connected to thedrive block 163, the piston 115 of the illustrated embodiment ispreferably oriented so a longitudinal axis 179 of the piston 115 isparallel to the threaded shaft 151. A piston alignment groove 193 (FIG.5) is preferably formed in either or both of the opposing surfaces ofthe clamp 185 and the anvil 189 to facilitate mounting the piston 115 onthe piston mount 181 in the desired alignment. The piston mount 181secures a piston 115 to the drive block 163 so the piston is constrainedto move substantially in unison with the drive block. Notably, thepiston mount 181 of the illustrated embodiment facilitates rapidreplacement of a piston with a new piston in the event a piston isdamaged or if it is desired to change the diameter or othercharacteristics of the piston.

A container holder 195 is also secured to the frame 133. Theconfiguration of the container holder can be varied to suit variousdesigns of the containers (e.g., micropipettes) from which the cellularpellet is to be extruded. In the embodiment shown in the drawings, forexample, the container holder 195 comprises a two part housing 203secured to the frame (e.g., by screws or other conventional fasteners211). Referring to FIG. 4, the housing 203 defines a cavity 207 sizedand shaped (e.g., generally cylindrically) for holding the container117. When a container 117 is loaded in the holder 195, one end of apiston 115 can be inserted into the container through a rear opening 213of the container and the other end of the piston can be secured to thedrive block 163. Preferably, the diameter of the piston 115 is selectedso there is a snug fit between the outside diameter of the piston and aninside diameter of the container 117.

An orifice 119 (FIG. 6) is formed at the end of the container holder195. The orifice 119 is preferably aligned with the groove 193 in thepiston mount 181, axis 179 of the piston 115, and the opening 213 at therear of the container 117. More preferably, a line extending from theorifice 119 to any of the groove 193 in the piston mount 181, axis 179of the piston 115, and the opening 213 at the rear of the container 117is parallel to the axis 157 of the threaded shaft 151. The orifice 119is an opening 215 at the front end of the container 117 as shown in FIG.4. However, the orifice could be formed in other ways (e.g., by the endof a through-hole from a location adjacent the opening 215 at front endof the container 117 to the exterior of the housing) without departingfrom the scope of the invention. The orifice 119 preferably has acircular cross section. The diameter of the orifice is preferablybetween about 100 and about 600 microns, more preferably between about200 and 400 microns.

In addition to the cutting blade 123, the cutting system 105 comprisesan actuator 219 that is connected to the cutting blade 123 so that theactuator is operable to cause the cutting blade to reciprocate acrossthe orifice 119, preferably in close proximity to (e.g., sliding contactwith) the opening 215 at the front of the container 117. Variousactuators are suitable for this job, including for example pneumaticactuators, hydraulic actuators, and motor-driven cams. In the embodimentshown in the drawings, for example, the actuator is a solenoid actuator219 under the control of the control system 129.

Many different arrangements of gears and/or linkages can be used toconnect the actuator 219 to the cutting blade 123 without departing fromthe scope of the invention. In the illustrated embodiment, the solenoidactuator 219 is positioned adjacent (e.g., in contact with) one end of apivot arm 223, which is pivotally mounted to a pivot base 225 secured tothe frame 133 so that the actuator is operable to rotate the pivot arm.The other end of the pivot arm 223 comprises a cam 227. A cam follower231 is formed on a plate 235 which is slideably mounted on the frame133. In the illustrated embodiment, the plate 235 is slideably mountedon a plurality of guide posts 239 (FIG. 4) secured to one of the blocks137 of the frame 133. The plate 235 is preferably biased (e.g., by oneor more springs 245 axially compressed between the plate and the frame133) to move toward the cam 227.

A cutting blade holder 249 is secured to the plate 235. The cuttingblade holder 249 is operable to hold the cutting blade 123 (e.g., arazor blade) so its cutting edge is adjacent the orifice 119 throughwhich the pellet is to be extruded. In the embodiment shown in thedrawings, the blade holder 249 comprises a blade mounting assembly 251including two generally parallel plates 255 fastened together (e.g., byscrews 259 or other suitably fasteners). The cutting blade 123 can beplaced between the two plates 255 before they are fastened together sothat the blade is secured to the mounting assembly 253 when the platesare fastened together. When so mounted, the cutting blade 123 ispreferably flush against a sliding surface 261 formed on the side of thehousing centered around the orifice 119 with the orifice 119 andoriented so the cutting edge of the cutting blade is adjacent theorifice.

To use the apparatus 101 to make a plurality of cell aggregates that aresuitable for bio-ink particles, the container 117 (e.g., micropipette)containing a pellet comprising a plurality of cells, prepared asdescribe above for example, is loaded in the container holder 195 (FIG.4). A piston 115 is inserted into the rear opening 213 of the container117 and also secured to the drive block 163 via the piston mount 181.The motor 111 is activated to rotate the shaft 151 and thereby move thedrive block 163 away from the motor, which causes the piston to movethrough the container 117 toward the orifice 119. Accordingly, theextruding system 103 begins extruding the pellet through the orifice119.

The motor 111 is paused after a period of time (e.g., as determined by atiming system of the control system 129) to make the portion of thepellet that has been extruded through the orifice 119 stationary. Whilethe motor 111 is paused, the control system 129 causes the cuttingsystem 105 to cut the pellet adjacent the orifice 119 to thereby createa cell aggregate having a cylindrical shape. Preferably the controlsystem 129 directs the motor 111 to paused upon formation of anextrusion having a length that is about equal (e.g., within about 10-20microns of equal) to the diameter of the orifice 119 through which thepellet is being extruded to produce a cylindrical cell aggregate thathas a diameter that is about equal to its length.

In the illustrated embodiment, for example, the cutting system is in aninitial position as is depicted in FIG. 6. The control system 129 causesthe cutting system 105 to cut the pellet by energizing the actuator 219.The actuator 219 rotates the pivot arm 223 to cause the cam 227 to pushagainst the cam follower 231 to move the plate 235 against its bias.This causes the cutting edge of the cutting blade 123 to slide over thesliding surface 261 across the orifice 119, preferably within no morethan about 2 microns of the opening 215 at the front end of thecontainer 117, and more preferably flush against the front end of thecontainer. As the cutting edge of the blade 123 moves across the orifice119, it cuts (i.e., severs) the extruded part of the pellet (i.e., theextrudate) from the part of the pellet that is still in the container119, thereby producing a cylindrical cell aggregate, which falls into acollection container (not shown) under the cutting blade 123. At thispoint the cutting system 105 is in the state depicted in FIG. 7. Thecollection container can contain a growth/support medium that issuitable for the particular cells in the cell aggregates. The collectioncontainer can also be agitated (e.g., on a gyratory shaker) if desiredto produce spherical aggregates as described above.

After the cell aggregate has been cut, the control system 129de-energizes the solenoid actuator 219, thereby allowing the bias of theplate 235 to move the plate away from the block 137, which causes thecutting blade 123 to return to the position it was in before the controlsystem energized the solenoid. The movement of the plate 235 alsorotates the pivot arm 223 in the opposite direction until the pivot armis positioned as it was before the solenoid actuator moved it (FIG. 6).The motor 111 is restarted to resume extrusion of the pellet through theorifice 119. The process is repeated as long as desired or until thecontainer 117 is empty. Each cycle preferably lasts for a period rangingfrom about 2 to about 3 seconds, resulting in production of cellaggregates at a rate ranging from between about 20 to about 30aggregates per minute.

Control Unit

One embodiment of the control unit comprises a computer that containsinformation about the shape of a target organ and properties of one ormore matrices and also provides instructions for the dispensing unit.For example, the computer control unit can store the anatomical“blueprints” (i.e., map) of target organs and the information on thedesired biodegradable gels or polymers in relation to the bio-inkparticles used. The blueprint can be derived from digitized imagereconstruction of a natural organ or tissue. Imaging data can be derivedfrom various modalities including noninvasive scanning of the human bodyor a detailed 3D reconstruction of serial sections of specific organs.The control unit may be the same control unit used to control theextrusion system and cutting system of the apparatus, as describedabove, but separate control systems could be used for the process ofmaking the cell aggregates and the organ printing process withoutdeparting from the scope of the invention.

Dispensing Unit

In one exemplary embodiment, the dispensing unit is in electroniccommunication with and under the control of the control unit. Thedispensing unit comprises a plurality of dispensers separately holdingone or more compositions comprising bio-ink particles and one or moreselected biodegradable gels or polymers. The dispensing unit alsocomprises a dispensing platform (e.g., a temperature-controlledsubstrate onto which the bio-ink particles and gels or polymers aredeposited). The dispensing unit functions as a special purpose deliverydevice, capable of depositing bio-ink particles and biodegradable gelsor polymers onto the dispensing platform, according to the instructionsfrom the control unit.

Preferably, cell aggregates are loaded into one of the dispensers. Forexample, the cell aggregates can be aspirated into a dispenser (e.g.,micropipette) one at a time. The dispenser is preferably a cylindricalcontainer having an inner diameter that is about the same size as thediameter of the cell aggregates. As each cell aggregate is aspiratedinto the dispenser, a quantity of the suspension medium may also beaspirated into the dispenser. It is preferable to expel as much of thesuspension medium as possible before aspirating the next cell aggregate.The result of this process is a single-file stack of cell aggregateslined up in the container and a minimal amount of the support medium.This arrangement facilitates controlled deposition of single cellaggregates by the dispenser. There is a limited time to dispense thecell aggregates before they fuse to each other.

A variety of printing or dispensing devices can serve as the dispensingunit, such as jet-based cell printers, cell dispensers or bio-plotters.For example, the dispensing unit disclosed in U.S. patent applicationSer. No. 10/891,512 (Pub. No. 2004/0253365), the contents of which arehereby incorporated by reference, can readily be adapted for use in thepresent invention by re-dimensioning some of the dispensers (e.g.,cartridges) so they are suitably sized to contain and dispense acomposition comprising cell aggregates rather than individual cells. Aninstrument as described in the '512 application may be acquired fromSciperio, Inc. of Stillwater, Okla.

Maturation Unit

The maturation unit is a bioreactor that assures the proper post-processhandling of the resulting construct. The maturation unit, depending onthe complexity of the organ module, can comprise a simple incubator or aspecifically designed bioreactor adapted to the particular needs of aspecific organ printing process. This will depend in part on the type oftissue being produced as is well known to those skilled in the art.

Computer Modeling

The methods of the present invention can be used to reliability “print”a tissue having a desired structure. The invention can also facilitateselection of materials (e.g., cells and matrices) and other processvariables (e.g., embedding patterns) that are best suited for theprocess.

When bio-ink particles are embedded in a matrix, the structuralevolution of the cells depends on a number of variables includingadhesive forces between the cells, adhesive and cohesive forces betweenthe cells and the matrix, the characteristics of the matrix (includingcomposition and spatial structure), and the pattern by which the cellaggregates are embedded in the matrix. Information about these variablescan be used to run computer simulations to make useful predictions ofthe structural evolution. These predictions allow selection ofcombinations of biodegradable polymers or gels for use in forming thematrix, the particular bio-ink particles, and/or the embedding patternby which the bio-ink particles are to be embedded in the matrix to beoptimized for any specific organ printing process.

One embodiment of such a model considers the structural evolution of thecells comprising a plurality of cell aggregates that consistsessentially of a single type of cell, after they are embedded in auniform matrix. The model represents the cells and matrix elements asparticles occupying the nodes of a discrete lattice (e.g., a cubiclattice). It uses information about specific interactions (e.g., thestrength of adhesion or cohesion forces) between cells, cells and thematrix and between matrix volume elements. A three-dimensional model isconstructed (e.g., a numerical model suitable for use in a computersimulation), in which the sites of a lattice are occupied either bypoint-like cells or matrix volume elements. For a model based on a cubiclattice, the total interaction energy, E of the system is written as

$E = {\sum\limits_{{< r},{r^{\prime} >}}\; {J\left( {\sigma_{r},\sigma_{r^{\prime}}} \right)}}$

where r and r′ label lattice sites, and <r,r′>signifies summation overneighboring sites, each pair counted once. First, second and thirdnearest neighbors are included, and it is assumed that a cell interactswith the same strength with all the 26 cells it comes into contact with.(26 is the total number of first, second and third nearest neighbors ofa given site in a cubic lattice.) To specify occupancy, a spin value, σ,is assigned to each lattice site with values 0 for a “matrix particle”and 1 for a cell. The interaction energy of two neighbors,J(σ_(r),σ_(r)), may take either of the values J(0,0)=−ε_(gg),J(1,1)=−ε_(cc) or J(0,1)=J(1,0)=−ε_(cg). The positive parameters ε_(cc),ε_(gg) and ε_(cg) account for contact interaction strengths forcell-cell, matrix-matrix and cell-matrix pairs, respectively. Morespecifically, these are mechanical works needed to disrupt thecorresponding bonds. (Note that ε_(cc) and ε_(gg) are works of cohesion,whereas ε_(cg) is work of adhesion per bond).

The strength of cell-cell interaction may be determined experimentallyeither directly or by measuring the tissue surface tension. Thecell-matrix interaction is tunable by biochemical methods or by theconcentration of a polymer forming the matrix. The matrix-matrix “bondenergy” is an effective measure of matrix filament density, interactionand stiffness. It is determined by the specific chemistry of the matrix.For example, the matrix-matrix interaction can be evaluated by measuringthe viscoelastic moduli (e.g., the loss and/or storage modulus) of thematrix. The cell-matrix interaction can be evaluated in the same way, bymeasuring how the viscoelastic moduli of the matrix is affected byintroduction of cells into the matrix.

The foregoing energy equation may be rewritten by separating interfacialand bulk terms in the sum:

E=γ _(cg) B _(cg) +const.

where B_(cg) is the total number of cell-matrix bonds, andγ_(cg)=(ε_(cc)+ε_(gg))/2−ε_(cg) is proportional to the cell-matrixinterfacial tension. The remaining terms in E do not change as thecellular pattern evolves.

The structural evolution of the system can be predicted using MonteCarlo simulations, relying on a random number generator. One cell on theaggregate-matrix interface is selected at random and exchanged with arandomly selected adjacent matrix volume element. The correspondingchange in total interaction energy, ΔE, is calculated and the newconfiguration accepted with a probability P=1 if ΔE≦0 or P=exp(−βΔE)ifΔE>0. β=1/E_(T), is the inverse of the average biological fluctuationenergy E_(T), analogous to the thermal fluctuation energy, k_(B)T(k_(B)-Boltzmann's constant, T-absolute temperature). In statisticalmechanics the thermal fluctuation energy characterizes thermal agitationin a system of atoms or molecules. In the case of cells, it is a measureof the spontaneous, cytoskeleton driven motion of cells, able to breakadhesive bonds between neighbors via membrane ruffling, or moregenerally, via membrane protrusive activity (e.g., filopodialextensions). By definition, a Monte Carlo step (MCS) is completed wheneach cell at the cellular material-matrix interface has been given theopportunity to move once. During each MCS the interfacial sites areselected in random order. The matrix boundary is treated as a fixedphysical limit of the system, and cells are constrained to move withinthe matrix.

Depending on the properties of the polymers or gels used to form thematrix, the characteristics of the bio-ink particles, and the embeddingpattern, the bio-ink particles represented in the model may or may notevolve into a desired structure. In some cases the constituent cellsdisperse into the surrounding matrix. In other cases, the constituentcells may quickly collapse into an undesired structure.

Some structures of cells embedded in a matrix correspond to long-livedstructures, the energies of which are approximately constant for a largenumber of MCS (See FIG. 11 discussed later herein in connection with theexamples). They correspond to the structure of the model associated withplateaus in the plot of the total interaction energy vs. the number ofMCS. The Monte-Carlo method does not account for passage of time per se,but the number of MCS is generally correlated with passage of time sothat structures that are relatively stable for many MCS can bedetermined to be long-lived. Long-lived structures are important fortissue engineering, because they suggest that the physical systemrepresented by the model can evolve into the long-lived structureindicated by the model. Then the fused aggregates can be separated fromthe matrix (e.g., by degrading the matrix by exposure to heat, light orany other stimulus effective to degrade the particular matrix) to obtaina tissue having the same general structure as the long-lived structureby the model.

The model can be generalized as necessary to simulate other physicalsystems involving the structural evolution of the cells of a pluralityof cell aggregates embedded in a matrix. Thus, the computer simulationcan readily be adapted for use in predicting structural evolution formore complicated organ printing processes using the principles set forthabove. For example, the matrix can comprise a plurality of differentsubstances having distinct properties and/or the cell aggregates maycomprise different cell types having different characteristicinteractions with the matrix (or the various components thereof). Toaccount for these additional variables, the total interaction energyequation is simply revised to account for the various different kinds ofinteraction forces at work in the system.

Consider, for example, that the physical system represented by the modelis a cylindrical congregation of cell aggregates comprising cells of twotypes (e.g., multi-color bio-ink; wherein the first type are epithelialcells are the second type are smooth muscle cells) embedded in a matrixcomprising a volume of a first substance, which is supportive of thecells of the first type, forming a core through the cell aggregates anda volume of a second substance, which is supportive of the cells of thesecond type, surrounding the cells aggregates and the volume of thefirst substance. Notably, this physical system could represent an organprinting process for production of a tubular organ wherein the firsttype of cells form the lumen and the second type of cells form smoothmuscle surrounding the lumen. The energy equation can be modified toaccount for the various interactions in the total energy equationincluding: (1) cells of the first type with cells of the first type; (2)cells of the first type with cells of the second type; (3) cells of thefirst type with the first substance; (4) cells of the first type withthe second substance; (5) cells of the second type with the cells of thesecond type; (6) cells of the second type with the first substance; (7)cells of the second type with the second substance; (8) volume elementsof the first substance with other volume elements of the firstsubstance; (9) volume elements of the second substance with other volumeelements of the second substance and (10) volume elements of the firstsubstance with other volume elements of the second substance.

By executing computer simulations to predict how the structuralevolution of constituent cells of a plurality of cell aggregatesembedded in a matrix according to an embedding pattern changes asprocess variables (including, for example, the type of cells used toform the cell aggregates, the substances used to form the matrix, andthe size, shape and configuration of the embedding pattern) change, onecan determine whether a particular combination of cell aggregates,matrix, and embedding pattern is likely to allow a desired tissuestructure to be produced without running an actual experiment on a realphysical system. Further, an understanding of how the cellular patternevolves in a series of modeled experiments based on differing processvariables can be used to optimize process variables to obtain a betterresult.

For example, the modeling methods may indicate that results wouldimprove if the adhesion of the cells could be modified by some factor.Similarly, the model methods may indicate that the size of geometricelements of the embedding pattern need to be adjusted (e.g., to accountfor contraction of the structure). Likewise, the modeling methods mayindicate that using a matrix having different properties would lead tomore desirable results.

Thus, in one embodiment of the invention, the methods described aboveare used to predict how various matrix candidates will perform when aplurality of cell aggregates are embedded in the candidate matricesaccording to a specified embedding pattern. One of the candidatematrices exhibiting the best results in the modeling can be selected foruse in producing a plurality of fused cell aggregates having a desiredthree-dimensional structure. In another embodiment of the invention themodeling methods are used to predict how various candidate cell typeswill perform when a plurality of cell aggregates comprising candidatecells are embedded in a specified matrix according to a specifiedembedding pattern. One of the cell candidates exhibiting the bestresults in the, modeling can be selected for use in producing aplurality of fused cell aggregates having a desired three-dimensionalstructure. In still another embodiment of the invention the modelingmethods described above are used to predict the performance of variouscandidate embedding patterns when a plurality of cell aggregates areembedded in a specified matrix in accordance with the candidateembedding patterns. One of the candidate embedding patterns exhibitingthe best results in the modeling can be selected for use in producing aplurality of fused cell aggregates having a desired three-dimensionalstructure. Moreover, the modeling methods can be adapted to predictperformance of various candidate combinations of cells, matrices, andembedding patterns. One of the combinations of cells, matrices, andembedding patterns exhibiting the best results in the modeling can beselected for use in producing a plurality of fused cell aggregateshaving a desired three-dimensional structure.

Method of Organ Printing

According to one embodiment of a method of the present invention a layerof a matrix (e.g., a solidifying biodegradable gel or polymer havingknown properties) is deposited on a substrate. The layer of matrix maycomprise a uniform layer of a single substance or it may comprisemultiple substances deposited to form the layer according to apre-determined pattern. A plurality of cell aggregates are embedded inthe layer according to a pre-determined pattern. Optionally, the matrixand the cell aggregates are deposited simultaneously. The thickness ofthe layer is preferably about the same as the diameter of the cellaggregates. The cell aggregates can comprise cell aggregates thatcontain multiple cell types (e.g., a mixture of at least two types ofcells). The cell aggregates can also comprise one cell aggregate cellsconsisting essentially of a single type of cells (e.g., epithelialcells) and another cell aggregate consisting essentially of a singletype of cells (e.g., connective tissue forming cells) having differentproperties. It is understood that some cell aggregates, such as thoseconsisting essentially of connective tissue forming cells, can include amatrix substance (i.e., an extra-cellular matrix) and still beconsidered to consist essentially of a single type of cells.

These two steps are then repeated as necessary to embed cell aggregatesin the matrix, layer-by-layer, according to an embedding pattern that ispredicted to result in a cellular pattern evolving into a plurality offused cell aggregates forming a desired three-dimensional structure. Theembedded cell aggregates are placed in the maturation unit, in whichpattern evolution takes place resulting in the cell aggregates fusingand forming the desired tissue or organ geometry. If the cell aggregatescomprise multiple cell types, this can result in the cells segregatinginto different distinct populations of cells. When the cells haveevolved into a tissue structure having the desired structure, the tissuestructure can be separated from the matrix via standard procedures formelting or dissolving the matrix.

Using bio-ink particles as building blocks instead of isolated cells hasseveral advantages. First, using bio-ink particles can significantlyreduce the processing (actual printing) time to achieve the desiredstructure. Reduced processing time enhances cell survival. Second, usinga plurality of cells (e.g., multitude of cells) in a more physiologicalenvironment (i.e., allowing the cells to adhere to each other or theaggregate matrix) provides better conditions to achieve a highconcentration of healthy cells, which is difficult to achieve by othermethods. Third, because bio-ink particles may contain several cell typesand a pre-built internal structure, it is easier to engineer desiredstructures and considerable time can be saved during post-process tissueand organ maturation. Finally, the mechanical hardship involved in thedispensing process is less damaging for bio-ink particles than forindividual cells.

EXAMPLES Example 1 Cell Aggregate Preparation

Chinese Hamster Ovary (CHO) cells, transfected with N-cadherin (courtesyof A. Bershadsky, Weizmann Institute, Rehovot, Israel), were infectedwith histone binding H2B-YFP retrovirus (provided by R. D. Lansford,Beckman Institute at California Institute of Technology). Confluent cellcultures (3-4×10⁶ cells/75 cm² TC dish) grown in Dulbecco's ModifiedEagle Medium (DMEM, Gibco BRL Grand Island, N.Y.; supplemented with 10%FES (US Biotechnologies, Parkerford, Pa.), 10 μg/ml of penicillin,streptomycin, gentamicin, kanamycin, 400 μg/ml geneticin), were washedtwice with Hanks' Balanced Salt Solution (HBSS) containing 2 mM CaCl₂,then treated for 10 minutes with trypsin 0.1% (diluted from 2.5% stock,Gibco BRL, Grand Island, N.Y.). Depleted cells were centrifuged at 2500RPM for 4 minutes. The resulting pellet was transferred into capillarymicropipettes of 500 μm diameter and incubated at 37° C. with 5% CO₂ for10 minutes. The firm cylinders of cells removed from the pipettes werecut into fragments (500 μm height) using an automated cutting device,then incubated in 10-ml tissue culture flasks (Bellco Glass, Vineland,N.J.) with 3 ml DMEM on a gyratory shaker at 120 RPM with 5% CO₂ at 37°C. for 4 hours. This procedure provided substantially sphericalaggregates of substantially uniform size (˜500 μm diameter). Thisprocedure has been used to produce substantially spherical aggregates ofsubstantially uniform size having different diameters as well.

Example 2 Formation of a Fused Ring Structure

The cell aggregates prepared in Example 1 were used to engineer a threedimensional tissue construct by either “manually printing” or usingcomputer controlled delivery devices to print (i.e., embedding) theaggregates into biocompatible gels in a predetermined pattern.

NeuroGel™ (a biocompatible porous poly[N-(hydroxypropyl)methacrylamide]hydrogel) disks of 10 mm diameter and 2 mm thickness, containing RGDfragments (provided by Stephane Woerly, Organogel Canada, Quebec) werewashed three times with DMEM to eliminate the storage medium. A 0.5 mmwide, 0.5 mm deep circular groove was cut into a disk. Ten aggregateswere placed (either manually or by printing using a device as describedherein) contiguously in the groove to form a closed circle. The groovewas refilled with the gel to completely embed the aggregates. Thisstructure was incubated at 37° C., 5% CO₂ for 72 hours in a tissueculture dish containing 10 ml DMEM, washed with PBS, and finallyembedded in Tissue-Tek® OCT Compound (Electron Microscopy Sciences, FortWashington, Pa.). The structure was slowly cooled (1° C./min) to −20° C.in a Nalgene freezing container (Nalgene Labware, Rochester, N.Y.). Tovisualize aggregate fusion, at the end of the experiment, cryosectioningwas performed with a Reichert 2800N Frigocut cryotome (Reichert Jung,Arnsberg, Germany), yielding 10-16 micron thin slices mounted onmicroscope slides. Slices were examined on an Olympus IX-70 invertedmicroscope with fluorescent attachment at 4× magnification. The resultsare shown in FIGS. 8C and 8D.

To tune the strength of cell-gel interaction, further fusion experimentswere conducted using rat-tail collagen type I (Sigma-Aldrich, St. Louis,Mo.). The collagen was dissolved in 1N acetic acid, and then treatedwith Ham's F12 medium with sodium bicarbonate. At room temperature thismixture gels in a few minutes depending on concentration. Thegel-aggregate structure was achieved by creating a ring of tenaggregates placed contiguously on the top of a previously (almost)solidified collagen layer, then covering the aggregates with liquidcollagen that embedded the aggregates after gelation. These samples wereincubated under the same conditions as described above. This process wasperformed using 1.0, 1.2 and 1.7 mg/ml collagen. The resulting sampleswere transparent, thus it was possible to follow pattern (i.e., toroid)evolution in time by phase contrast and fluorescent microscopy. Cellsurvivability was checked with Trypan Blue (Invitrogen, Carlsbad,Calif.) at the end of each fusion experiment. A minimal number ofuniformly distributed dead cells were found. The results are shown inFIGS. 8E-8J.

Results

The experiments were carried out by the system described herein. Theexperiments were performed with fixed cell-cell adhesion and varying gelproperties, by depositing or printing N-cadherin transfected CHO cellaggregates into Neurogel™ disks and 1.0, 1.2, and 1.7 mg/ml. collagen.As indicated by the teachings of the invention, the ability ofaggregates to fuse depends on the mutual properties of the cellaggregates and gel or polymers, as expressed by the parameterγ_(cg)/E_(T) in the model. The transfected CHO cells' adhesiveproperties were quantitatively assessed, by measuring aggregate surfacetension using techniques such as those described in Foty, et al., Dev.Biol. 278:255-63 (2005). The relative importance of cell-cell andcell-matrix interactions has also been investigated quantitatively. See,e.g., P. L. Ryan, et al., PNAS 98, 4323-4327 (2001).

FIGS. 8A-8L show initial (FIGS. 8A, 8C, 8E, 8G, 8I, and 8K) and final(FIGS. 8B, 8D, 8F, 8H, 8J, and 8L) cell aggregate configurations in bothsimulations and in the experiments using the various biocompatible gels.FIGS. 8A-8B and 8K-8L correspond to simulations that were run using themodel described herein, with γ_(cg)/E_(T)=0.9 and γ_(cg)/E_(T)=0.25,respectively. The ten aggregates, each containing 925 cells were onecell diameter from each other in the starting configurations. The finalconfigurations were reached after 25,000 and 50,000 MCS, respectively.

The results of the simulation show that evolution of the cellularpattern is governed by a single parameter, γ_(cg)/E_(T), which for cellswith specific adhesion apparatus, is controlled by the chemistry of thegel. The theoretical analysis shows that, once gel properties areappropriately tuned, efficient fusion of adjacent aggregates takesplace. For small γ_(cg)/E_(T) (=0.25 in panels K-L) cells can spread inthe bulk of the gel and the pattern evolves towards its lowest energystate, being a sphere. Under optimal cell-gel interface properties, forexample, as depicted in FIGS. 8A-8B (γ_(cg)/E_(T)=0.9), fusion ofaggregates results in a 3-dimensional toroidal structure.

FIGS. 8C-8D, 8E-8F, 8G-8H and 8I-8J correspond to the experimentalresults using the CHO cell aggregates embedded in a Neurogel™ disk andin collagen gels of concentration 1.0, 1.2, and 1.7 mg/ml respectively.FIGS. 8I-8J show that collagen at concentration of 1.7 mg/ml isanalogous to a permissive scaffold with small γ_(cg)/E_(T). Collagen atconcentration of 1.0 and 1.2 mg/ml match more the definition of thenon-permissive gel with high γ_(cg)/E_(T). These gels favor much less(collagen), or not at all (neurogel), the dispersion of the cells intothe scaffold, thus facilitating fusion.

FIG. 9A shows the time variation of the boundary between two adjacentaggregates in the 1.0 mg/ml collagen gel (see FIGS. 9B and 9C). Ameasure of fusion is the instantaneous value of the angle formed by thetwo aggregates. As aggregates coalesce, the angle between the tangentsto their boundaries (drawn from the point where they join) approaches180°. The curve in FIG. 10 is an exponential fit to the data in the formC[1-exp(−t/τ_(cc))] (C-constant), with τ_(cc)≈23 h. Here the quantityτ_(cc) defines a time scale of aggregate fusion.

Example 3 Identification of a Long-Lived Structure

Shapes corresponding to long-lived structures (e.g., toroidalstructures), may be identified from plateaus in the plot of the totalinteraction energy vs. MCS. This is illustrated in more detail in thesimulation shown in FIG. 11A and FIG. 11B (γ_(cg)/E_(T)=1.1), where theinitial state progresses towards a long-lived toroidal configuration,whose energy is essentially unchanged in the entire interval between 10⁴and 6×10⁴ Monte Carlo steps (MCS) (FIG. 11A). Eventually the toroidbecomes unstable, and at about 10⁵ MCS it ruptures (FIG. 11B).Subsequent massive rearrangements lead to a pronounced energy decreasewhile the system evolves into three rounded aggregates. These remainstable for a long time because large spatial separations hinder theirfusion into a single spheroid.

Once a structure reaches the long-lived state, it can be stabilized bydissolving the supporting gel or polymer. In the simulations thiscorresponds to increasing the value of γ_(cg)/E_(T). Indeed, if in thesimulation shown in FIG. 11A this quantity is changed to γ_(cg)/E_(T)=2anywhere in the plateau region, the energy remains constant as long asthe simulation is run.

Example 4 Model Fusion of a Ring Structure and Formation of a CellularTube

Fusion of a ring structure and formation of a cellular tube weresimulated using the models described herein, to demonstrate the abilityof these models to predict aggregate fusion and structure formationbased on varying parameters.

Modeling Biological Structure Formation Based on Tissue Liquidity

The fusion of contiguously arranged aggregates into a ring structure wassimulated for varying interfacial tensions between the embedding gel andcellular material using the model described herein. Referring to FIG.12, ten aggregates 1201 of 4169 cells each were contiguously arrangedinto an initial ring pattern 1202. Simulations were then run on theinitial ring structure using varying values for γ_(cg)/E_(T). Theresults 1203, 1204, 1205 were recorded photographically.

The results of the simulations indicate that the ability of the ring ofaggregates to fuse depends on the interfacial tension, γ, between theembedding gel (not shown) and the cellular material. The structure 1203was the resulted when γ_(cg)/E_(T)=10 (analogous to agarose gel). E_(T)is the average biological fluctuation energy, which is analogous to thethermal energy in true liquids. E_(T) is a measure of the spontaneous,cytoskeleton driven motion of cells (see Mombach, et al., Phys. Rev.Lett. 75, 2244-2247 (1995)), and its value depends on cell type. Thestructure 1204 was the result when γ_(cg)/E_(T)=0.25 (analogous to 1.7mg/ml collagen gel). The structure 1205 was the result whenγ_(cg)/E_(T)=0.9 (analogous to 1.0 mg/ml collagen gel).

The results indicate that if γ is high (e.g., γ_(cg)/E_(T)=10), cellsare unable to move and the initial configuration remains practicallyfrozen (i.e., no fusion takes place) as indicated by the structure 1203.If the interfacial tension is small (e.g., γ_(cg)/E_(T)=0.25) cells caneasily migrate into the gel and the system is able to reach its truelowest energy state, a single spherical configuration, as indicated bystructure 1204. Finally, for intermediate values of γ (e.g.,γ_(cg)/E_(T)=0.9) a long-lived fused ring forms, as indicated bystructure 1205. During evolution of the long-lived structure 1205, thenumber of cell-matrix bonds, B_(cg) remains practically unchanged formany thousands of Monte Carlo steps (MCS), as evidenced by the curve(500) in FIG. 14. In the present model B_(cg) is a quantity related tothe overall energy and proportional to the surface area of thestructure. Changes in the slope of B_(cg) vs. MCS correspond tomodifications in construct topology. In the studied range of MCS theseare more frequent for γ_(cg)/E_(T)=0.25, as evidenced by the curve (501)in FIG. 14, due to faster pattern evolution. The final configurationsshown in FIGS. 12(3), (4) and (5) were reached in 2.5×10⁵ MCS.

Model Cellular Tube Formation

The formation of a cellular tube from an initial state comprising 15layers of cellular rings was simulated using the model described herein(FIG. 13). Ten aggregates comprising 257 cells each were placedcontiguously along circles to form a ring. A second ring layer, againcomprising ten contiguous aggregates of 257 cells each, was closelypacked in the vertical direction on the first ring layer, so that eachaggregate in the second ring touched two other aggregates from below.This was repeated until a structure 1301 comprising 15 layers of ringswas simulated. Simulations for the resulting 15 layer structure 1301were then run, using varying values for γ_(cg)/E_(T). The resultingstructures 1302, 1303 were recorded.

When γ_(cg)/E_(T)=0.4, the 15 layer structure 1301 breaks up into twospheroids 1302 and remains in this state for a prolonged period of time,as evidenced by the curve (502) in FIG. 15. The relatively large spatialseparation between the spheres makes their fusion into a singlestructure difficult. When γ_(cg)/E_(T)=2, after 2×10⁵ MCS the 15 layerstructure 1301 fuses to form a tube 1303, which is a robust, long-livedstructure, as evidenced by the curve (503) in FIG. 15.

Example 5 Formation of a Fused Ring and Fused Tube Structure UsingFluorescently Labeled Cell Aggregates Formation of a Fused RingStructure

CHO cells as described in Example 1 were fluorescently labeled with twodifferent membrane intercalating dyes of different color, in order tobetter illustrate cell aggregate fusion process.

Prior to aggregate formation, the CHO cells were stained with eitherPKH26 Red Fluorescent General Cell Linker or PKH2 Green FluorescentGeneral Cell Linker (Sigma, St. Louis Mo.) as recommended by themanufacturer. The procedure described in Example 1 was repeated usingthese fluorescently labeled CHO cells, to form fluorescently labeled CHOcell aggregates of approximately 480 microns in diameter. The cellaggregates 1601 comprised about 40,000 cells per aggregate.

Ten of the fluorescently labeled cell aggregates were contiguouslyarranged (either manually or printed using the device described herein)to form a closed circle 1602 on either agarose gel, 1.7 mg/ml collagen,or 1.0 mg/ml collagen, as prepared in Example 2. The aggregates werearranged in an alternating ring structure 1602 so that aggregateslabeled with PKH2 Green dye alternated with aggregates labeled withPKH26 Red dye, in order to better visualize fusion. The resultingstructure was incubated at 37° C., and then cooled to room temperature.Aggregate fusion was then detected using confocal microscopy.

After 80 hours of incubation, the final configurations 1603 (agarosegel), 1604 (1.7 mg/ml collagen), and 1605 (1.0 mg/ml collagen) wererecorded photographically. The boundaries 1606 of neighboring aggregatesand the embedding gel when 1.0 mg/ml collagen is used were also recordedphotographically. An enlarged view 1607 (at 60× magnification) of theinterior of the contact region between aggregates when 1.0 mg/mlcollagen is used was also recorded photographically.

Formation of a Fused Tube Structure

Referring to FIGS. 17A-17I, the fluorescently labeled CHO cellaggregates described above were used to form a three dimensional fusedtube structure.

Ten of the fluorescently labeled cell aggregates were contiguouslyarranged (either manually or printed) to form a closed circle on 1.0mg/ml collagen, as prepared in Example 2. A second ring layer, againcomprising 10 fluorescently labeled cell aggregates contiguouslyarranged to form a closed circle (manually or by printing using a 3Dbioprinter) on the 1.0 mg/ml collagen gel, was closely stacked in thevertical direction on the first ring layer. This process was repeateduntil a structure comprising three layers of vertically stacked ringswas produced, each layer comprising cell aggregates fluorescentlylabeled with a different color dye (e.g., PKH2 Green or PKH26 Red). Theresulting structure was incubated at 37° C. and cooled to roomtemperature, as described above. Aggregate fusion was then detectedusing confocal microscopy.

The resulting structures are shown in FIGS. 17A-17I. FIGS. 17A-17C arebright field images of pattern evolution of three vertically closelypacked rings towards a hollow tube at 0 hours (FIG. 17A), 12 hours (FIG.17B), and 24 hours (FIG. 17C). FIGS. 17D-17I are fluorescent images ofthis process after 0 hours (FIG. 17D), 12 hours (FIG. 17E), 24 hours(FIG. 17F), 36 hours (FIG. 17G), 48 hours (FIG. 17H), and 60 hours (FIG.17I).

CONCLUSION

When introducing elements of the present invention or the preferredembodiments thereof, the articles “a”, “an”, “the”, and “said” areintended to mean that there are one or more of the elements. The terms“comprising”, “including,” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements.

In view of the above it will be seen that the several objects of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above constructions,compositions, and methods without departing from the scope of theinvention, it is intended that all matter contained in the abovedescription and shown in the accompanying drawings shall be interpretedas illustrative and not in a limiting sense.

1. A method of producing a plurality of fused aggregates forming adesired three-dimensional structure, the method comprising: depositing alayer of a matrix on a substrate; embedding a plurality of cellaggregates, each comprising a plurality of cells, in the layer of thematrix, the aggregates being arranged in a predetermined pattern;allowing at least one aggregate of said plurality of cell aggregates tofuse with at least one other aggregate of the plurality of cellaggregates to form the desired structure; and separating the structurefrom the matrix.
 2. The method of claim 1 wherein the layer of thematrix constitutes a first layer, the plurality of cell aggregatesconstitutes a first plurality of cell aggregates, and the predeterminedpattern constitutes a first predetermined pattern, the method furthercomprising the steps of: depositing a second layer of the matrix on thefirst layer; and embedding a second plurality of cell aggregates in thesecond layer, the second plurality of cell aggregates comprising aplurality of cells, the second plurality of cell aggregates beingarranged in a second predetermined pattern, and allowing at least onecell aggregate in the first plurality of cell aggregates to fuse with atleast one cell aggregate in the second plurality of cell aggregates. 3.The method of claim 2 wherein the first and second predeterminedpatterns are substantially the same, and wherein the second plurality ofcell aggregates is embedded in the second layer of the matrix inregistration with the first plurality of cell aggregates.
 4. The methodof claim 2 wherein the desired structure is a tube, the first and secondpredetermined patterns are both circular in shape, and the secondplurality of cell aggregates is embedded in the second layer of thematrix in registration with the first plurality of cell aggregates. 5.The method of claim 1 wherein the thickness of the layer of the matrixis about equal to the average diameter of the plurality of cellaggregates.
 6. The method of claim 1 wherein the cell aggregates aresubstantially spherical.
 7. The method of claim 1 wherein the cellaggregates are substantially uniform in size.
 8. The method of claim 1wherein the cell aggregates have an average size between about 100 andabout 600 microns.
 9. The method of claim 8 wherein no more than about10% percent of the cell aggregates deviate from said average size bymore than 5%.
 10. (canceled)
 11. The method of claim 1 wherein the cellaggregates consist essentially of cells of a single type.
 12. The methodof claim 1 wherein at least one of the cell aggregates comprises aplurality of cells of a first type and a plurality of cells of a secondtype that is different from the first type.
 13. The method of claim 12wherein said at least one cell aggregate comprises a mixture of saidcells of the first type and said cells of the second type and the methodfurther comprises the step of allowing at least some of the cells of thefirst type to segregate from at least some of the cells of the secondtype.
 14. The method of claim 13 wherein the cells of the first type areepithelial cells and the cells of the second type are connectivetissue-forming cells.
 15. The method of claim 1 wherein thepredetermined pattern comprises a ring.
 16. The method of claim 1wherein the matrix comprises a gel.
 17. The method of claim 1 whereinsaid plurality of cell aggregates includes at least one cell aggregateconsisting essentially of cells of a first type and at least one othercell aggregate consisting essentially of cells of a second typedifferent from the first type. 18-51. (canceled)
 52. A three-dimensionallayered structure comprising: at least one layer of a biocompatiblematrix; and a plurality of cell aggregates, each cell aggregatecomprising a plurality of living cells; wherein the cell aggregates areembedded in the at least one layer of biocompatible matrix in apredetermined pattern.
 53. The structure of claim 52 wherein the cellaggregates are substantially uniform in size and shape.
 54. Thestructure of claim 52 wherein the cell aggregates are cylindrical. 55.The structure of claim 54 wherein the cylindrical cell aggregates arefrom about 100 microns to about 600 microns in height.
 56. The structureof claim 52 wherein the cell aggregates are substantially spherical. 57.The structure of claim 56 wherein the substantially spherical cellaggregates are between about 100 and about 600 microns in diameter. 58.The structure of claim 52 wherein each cell aggregate comprises aplurality of living cells of a single cell type.
 59. The structure ofclaim 52 wherein each cell aggregate comprises a plurality of livingcells of a first cell type and a plurality of living cells of a secondcell type, the second cell type being different from the first celltype.
 60. The structure of claim 52 wherein the plurality of cellaggregates comprises a plurality of cell aggregates of a first cell typeand a plurality of cell aggregates of a second cell type, the secondcell type being different from the first cell type.
 61. The structure ofclaim 52 wherein the layer of biocompatible matrix is about 100 micronsto about 600 microns thick.
 62. The structure of claim 52 wherein thebiocompatible matrix is selected from the group consisting ofthermo-reversible gels, photo-sensitive gels, pH-sensitive gels, celltype specific gels, and combinations thereof.
 63. The structure of claim52 wherein the at least one layer of biocompatible matrix comprises atleast two different types of biocompatible matrices.
 64. The structureof claim 52 comprising: a first layer of the biocompatible matrix; and asecond layer of the biocompatible matrix deposited on the first layer;wherein the cell aggregates are embedded in the first layer and in thesecond layer in a predetermined pattern.
 65. The structure of claim 64further comprising at least one additional layer of the biocompatiblematrix deposited on the second layer, wherein the cell aggregates areembedded in the first layer, the second layer, and the at least oneadditional layer in a predetermined pattern.
 66. The structure of claim64 wherein the first layer comprises a type of biocompatible matrix thatis different from the type of biocompatible matrix in the second layer.